Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Nucleo-cytoplasmic shuttling of protein molecules is a basic biological process central to the regulation of gene expression (which underlies all aspects of development, morphogenesis, and signaling pathways in eukaryotic organisms). Furthermore, transport of proteins and protein-nucleic acid complexes in and out of the nucleus is an essential step in many host-pathogen interactions such as viral and bacterial infection. Nuclear traffic occurs exclusively through the nuclear pore complex (NPC). While small molecules (up to 40-60 kDa) diffuse through the NPC, nuclear import of larger molecules is mediated by specific Nuclear Localization Signal (NLS) sequences contained in the transported molecule (Garcia-Bustos et al. 1991; Dingwall 1991). Most NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (SEQ ID NO:3: PKKKRKV); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (SEQ ID NO:4: KRXXXXXXXXXXKKKL); and (iii) noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991).
Once in the nucleus, many proteins are transported back to the cytoplasm as an essential step in their biological function. For example, the Rev protein of human immunodeficiency virus type 1 (HIV-1): exits the nucleus, facilitating export of the unspliced viral RNA (Pollard and Malim 1998). Rev nuclear export is mediated by a specific Nuclear Export Signal (NES) consisting of the leucine-rich sequence, SEQ ID NO:5: LPPLERLTL, found also in proteins of other viruses (Dobbelstein et al. 1997). Also, numerous cellular proteins, such as I-KB and MAPKK, contain potential NES sequences which may regulate the biological activity of these proteins by controlling their nuclear export (Ullman et al. 1997).
The relatively small size of the NLS and NES sequences and, more importantly, the lack of clear and consistent consensus motifs in these signals, make it difficult to predict their presence in a given protein based solely on the analysis of its amino acid sequence. Furthermore, even if a consensus NLS or NES were found, it may not represent a functional signal. For example, β-glucuronidase (GUS), a commonly-used reporter enzyme which resides exclusively in the cell cytoplasm (Varagona et al. 1991; Citovsky et al. 1992), carries a perfect, albeit non-functional, bipartite NLS at its carboxy terminus. Thus, the only practical way to identify active NLS or NES signals is by microinjecting (Guralnick et al. 1996; Goldfarb et al. 1986; Kalderon et al. 1984) or expressing the protein of interest in eukaryotic cells (Varagona et al. 1991; Citovsky et al. 1992; Robbins et al. 1991; Roberts et al. 1987), heterokaryon formation (Michael et al. 1995), or using an in vitro transport system (Ossareh-Nazari et al. 1997; Schlenstedt et al. 1993; Newmeyer et al. 1988; Ballas and Citovsky 1997). Two major experimental approaches have been developed in this regard. In one approach, the protein of interest is labeled, microinjected into eukaryotic cells, and its intracellular localization determined. In another approach, the tested genes are fused to a reporter (β-galactosidase, green fluorescent protein, etc.), expressed in eukaryotic cells, and the localization of the resulting fusion protein determined. Both methods have serious technical disadvantages. The first approach is very labor-intensive and requires highly trained personnel experienced in protein purification, microinjection, and fluorescent or electron microscopy techniques. The second method is also very laborious, involving often elaborate procedures for genetic transformation of higher eukaryotic cells and microscopy observations. Since both of these procedures rely on physical intracellular localization of the protein, common artifacts such as perinuclear binding can present problems in analysis of results.
A need continues to exist, therefore, for a method for determining whether newly-cloned genes may encode a protein that localizes to or is exported from the cell nucleus.